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Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2019-10-23 to 2020-01-10
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2020
Report date:
2020

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
adopted 22 July 2010
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of study:
mouse local lymph node assay (LLNA)

Test material

Constituent 1
Chemical structure
Reference substance name:
5′-O-(4,4′-dimethoxytrityl)-2′-deoxythymidine-3′-O-[O-(2-cyanoethyl)-N,N′-diisopropylphosphoramidite]
EC Number:
685-410-3
Cas Number:
98796-51-1
Molecular formula:
C40H49N4O8P
IUPAC Name:
5′-O-(4,4′-dimethoxytrityl)-2′-deoxythymidine-3′-O-[O-(2-cyanoethyl)-N,N′-diisopropylphosphoramidite]
Test material form:
solid
Specific details on test material used for the study:
TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test item was placed into an appropriate container on a tared balance and DMF was added (weight per weight). The different test item concentrations were prepared individually. The preparations were made freshly before each dosing occasion.

In vivo test system

Test animals

Species:
mouse
Strain:
CBA/Ca
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Envigo RMS B.V., Inc, Postbus 6174, 5960 AD Horst, The Netherlands
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 8-9 weeks (pre-test), 8 weeks (main study)
- Weight at study initiation: 19.5-20.8 (pre-test), 16.5-20.7 g (main study)
- Housing: Animals were housed in a group, Makrolon Type II (pre-test) / III (main study) cages, with wire mesh top
- Diet: ad libitum, 2018C Teklad Global 18% protein rodent diet (certified)
- Water: ad libitum, tap water
- Acclimation period: At least 5 days prior to the start of dosing under test conditions after health examination. Only animals without any visible signs of illness were used for the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 (20-24)
- Humidity (%): approx. 45-65
- Photoperiod (hrs dark / hrs light): 12/12, artificial light (6.00 a.m.-6.00 p.m.)

Study design: in vivo (LLNA)

Vehicle:
dimethylformamide
Concentration:
5%, 10%, 25%, 50% (w/w)
No. of animals per dose:
- Pre-test: 2 female mice/group
- Main experiment: 5 female mice/group
Details on study design:
See "Any other information on materials and methods incl. tables" below.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
The mean values and standard deviations were calculated in the body weight tables. Where appropriate, the EC3 value were calculated according to the equation EC3 = (a-c) [(3-d)/(b-d)] + c, where EC3 is the estimated concentration of the test item required to produce a 3-fold increase in draining lymph node cell proliferative activity; (a, b) and (c, d) are respectively the co-ordinates of the two pair of data lying immediately above and below the S.I. value of 3 on the local lymph node assay dose response plot. All calculations conducted on the DPM values and the ear weights, were performed with a validated test script of “R”, a language and environment for statistical computing and graphics.
Within the program a statistical analysis conducted on the DPM values and the ear weights in order to assess whether the difference was statistically significant between the test item groups and negative control group. Statistical significance was set at the five per cent level (p< 0.05). Additionally, for the DPM values, the Dean-Dixon-Test and Grubb’s Test were used for identification of possible outliers. No outliers were detected in both statistical tests. However, both biological and statistical significance will be considered together.

Results and discussion

Positive control results:
α-hexyl cinnamaldehyde dissolved in acetone/olive oil (4+1 v/v) was used as a positive control. The positive control substance exceeded the stimulation index of 3 confirming the sensitivity and reliability of the experimental technique (see Table 2 in box "Any other information on results").

In vivo (LLNA)

Resultsopen allclose all
Key result
Parameter:
SI
Remarks:
mean of five animals
Value:
1.8
Test group / Remarks:
5%
Key result
Parameter:
SI
Remarks:
mean of five animals
Value:
1.4
Test group / Remarks:
10%
Key result
Parameter:
SI
Remarks:
mean of five animals
Value:
2
Test group / Remarks:
25%
Key result
Parameter:
SI
Remarks:
mean of five animals
Value:
1.8
Test group / Remarks:
50%
Cellular proliferation data / Observations:
DETAILS ON STIMULATION INDEX CALCULATION
- Please see Table 2 in box "Any other information on results incl. Tables".

EC3 CALCULATION
- The EC3 value could not be calculated, since all S.I.´s are below the threshold value of 3.

CLINICAL OBSERVATIONS:
- No deaths occured during the study period. No signs of systemic toxicity were observed during the study period. From day 1 to 6, the animals showed an erythema of the ear skin (Score 1 to 2). Additionally, scaly ears and fur loss was observed.

BODY WEIGHTS:
- The body weight of the animals, recorded prior to the first application and prior to treatment with 3HTdR, was within the range commonly recorded for animals of this strain and age.

Any other information on results incl. tables

Results of vehicle and dose selection:

Solubility test:

The maximum concentration of test item which could be technically used was a 50% solution in DMF.

Pre-test: Irritation and toxicity test:

Two mice were treated by (epidermal) topical application to the dorsal surface of each ear with test item concentrations of 25 and 50% once daily each on three consecutive days. At the tested concentrations the animals did not show any signs of systemic toxicity. From day 2 to 6, the animals showed an erythema of the ear skin (Score 1 to 2). Additionally, fur loss, stiffened ears, scaly ears and erythema of scalp were observed in the animal treated with the high dose of the test item. In the animal treated with the low dose, substance residuals and scaly ears were observed. A maximum increase in ear thickness of 10.4% (animal 1) and 7.5 % (animal 2) was observed. No excessive increase in ear weights was observed in both animals.

Results of the main study:

Table 2: Results of the positive control group

Test item (alpha-Hexylcinnamaldehyde) Concentration [%] Group Result Stimulation Index
- background -
- background -
0 1 1.00
5 2 1.68
10 3 1.78
25 4 8.19

Table 3: Results of the main experiment

Group calculation
Test item (DMT-dT-Phosphoramiditite) concentration [%] Group Number Animal Number DPM values measured DPM−BG per animal (2 lymph nodes) Stimulation Index Mean DPM per animal (2 lymph nodes) standard deviation Stimulation Index
- - background 1 13 - - - - -
- - background 2 21 - - - - -
Vehicle Control Group (DMF) 1 1 1457 1440 - 2378.0 709.0 1.0
1 2 2352 2335 -
1 3 2588 2571 -
1 4 3414 3397 -
1 5 2164 2147 -
5% DMT-dT-Phosphoramidite 2 6 3310 3293 1.4 4323.0 1598.8 1.8
2 7 2841 2824 1.2
2 8 3492 3475 1.5
2 9 6526 6509 2.7
2 10 5531 5514 2.3
10% DMT-dT-Phosphoramidite 3 11 1863 1846 0.8 3287.8 1085.6 1.4
3 12 3225 3208 1.3
3 13 2798 2781 1.2
3 14 4716 4699 2.0
3 15 3922 3905 1.6
25% DMT-dT-Phosphoramidite 4 16 2049 2032 0.9 4849.0 1867.8 2.0
4 17 4839 4822 2.0
4 18 4394 4377 1.8
4 19 6138 6121 2.6
4 20 6910 6893 2.9
50% DMT-dT-Phosphoramidite 5 21 2528 2511 1.1 4270.4 1302.9 1.8
5 22 5766 5749 2.4
5 23 3460 3443 1.4
5 24 5165 5148 2.2
5 25 4518 4501 1.9

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
In conclusion, in a mouse local lymph node assay, the test item DMT-dT-Phosphoramidite is not to be considered a skin sensitizer under the test conditions of this study.
Executive summary:

In a dermal sensitization study conducted according to OECD 429, five young adult female CBA/CaOlaHsd mice per dose group were dermally exposed to DMT-dT-Phosphoramidite in DMF at concentrations of 5%, 10% and 25% and 50% (w/w) by topical application to the dorsum of each ear for three consecutive days and observed until day 6 in a local lymph node assay (LLNA). α-hexyl cinnamaldehyde dissolved in acetone/olive oil (4+1 v/v) was used as a positive control. The positive control substance exceeded the stimulation index of 3 at the highest dose tested (25%).

No signs of systemic toxicity were observed during the study period. From day 1 to 6, the animals showed an erythema of the ear skin (Score 1 to 2). Additionally, scaly ears and fur loss was observed.

No mortality or effects on body weight occurred during the study. None of the four tested concentrations exceeded a stimulation index of 3 (1.8 (5%), 1.4 (10%) and 2.0 (25%) and 1.8 (50%)). As a consequence, an EC3 value could not be calculated. In this study, DMT-dT-Phosphoramidite is not a dermal sensitizer.